Although kidney stones have affected mankind for as long as history has been recorded, they continue to present a considerable public health concern, and the prevalence of kidney stone disease has experienced an upswing in the past century. Recurrent kidney stone formers frequently lack any metabolic abnormalities, with the exception of an increase in urine calcium. In the kidney, the proximal tubule (PT) is responsible for the reabsorption of approximately two-thirds of filtered calcium. Evidence that PT calcium reabsorption occurs through a passive and paracellular route is abundant, although the precise mechanism by which this occurs is not known. The selectivity of paracellular permeability is determined in large part by claudins, a family of proteins found at the tight junction between epithelial cells. Claudin-2 is a cation-selective claudin that is expressed in leaky epithelia, including the PT and gastrointestinal tract. Claudin-2 knockout (Cldn-2 KO) mice have increased fractional calcium excretion compared with wild type (WT) mice. The cause of this increase in calcium excretion has not been investigated carefully. We hypothesize that claudin-2 mediates paracellular calcium reabsorption in the PT. For our first aim, we plan to assess the calcium wasting phenotype in Cldn-2 KO mice. We also provide evidence that Cldn-2 KO mice develop nephrocalcinosis, and we propose studies to investigate this relationship further. Extracellular fluid (ECF) volume contraction reduces urine calcium by causing an increase in PT calcium reabsorption. To test the hypothesis that claudin-2 contributes to PT calcium reabsorption, specifically, we propose experiments measuring urine calcium in WT and Cldn-2 KO mice following induction of ECF volume contraction. Heteromeric claudin interactions may play a significant role in the permeability properties of epithelial tissues. In the PT, the most abundanty expressed claudins appear to be claudin-2, claudin-3, and claudin-10a. Our second aim will determine whether close interaction of PT claudins occurs, in vitro and in situ, using proximity ligation assays. In addition, we propose measurement of calcium permeability in cells overexpressing PT claudins, including investigation of a human SNP of claudin-2 associated with low urine calcium. Evidence is abundant that claudin- 2 expression is increased in renal epithelial cells by MEK inhibition, in vitro. Our final aim examines the effect of MEK inhibition, n vivo, on renal claudin-2 expression and urine calcium excretion. The broad, long term objective of this research is to elucidate the role of claudin-2 in PT calcium reabsorption, including its potential as a therapeutic target for reduction of urine calcium and prevention of kidney stone formation in recurrent stone formers.